利用五特征转基因小鼠制备全人源血管内皮生长因子165单克隆抗体

Generation of fully human anti-vascular endothelial growth factor 165 monoclonal antibody with antitumor efficacy from transgenic five-feature mice

目的:利用转入人免疫球蛋白基因的五特征小鼠制备全人源血管内皮生长因子165(VEGF165)单克隆抗体,并初步探讨该单抗的抗肿瘤活性。方法:采用常规杂交瘤细胞融合技术,利用间接ELISA效价测定方法筛选能稳定分泌单抗的杂交瘤细胞株;比较了五特征小鼠和BALB/c小鼠的免疫效果;亲和色谱法从腹水中纯化单抗;利用分泌VEGF的人膀胱癌细胞系T24细胞考察单抗的体外抗肿瘤活性。结果:成功获得了4株能稳定分泌表达单抗的杂交瘤细胞株(V2,V50,V71,V75),建立了间接ELISA效价测定方法,发现同样方法免疫的BALB/c鼠血清效价约为五特征小鼠的10倍。采用甘露糖结合蛋白(MBP)亲和色谱柱从腹水中获得了纯化的单抗样品,经SDS-PAGE和Western blotting检测,证实该单抗为全人源VEGF165IgM单抗。体外抗肿瘤活性结果显示,V2、V75单抗对人类膀胱癌细胞系T24细胞的增殖有较好的抑制作用。结论:可利用五特征小鼠制备全人源抗VEGF单抗,该单抗具有潜在的抗肿瘤活性。

Aim： To prepare a fully human anti-VEGFl65（ vascular endothelial growth factor 165） monoclonal antibody with antitumor activity from five-feature mice which express human immunoglobin loci. Methods： A routine method for the generation of monoclonal antibodies（mAbs） against the human VEGF165 was developed. The immunizing effect between five-feature mice and BALB/c was observed and the mAb was purified through MBP IgM affinity chromatography. The effect of mAbs on antitumor was tested in vitro by T24 cell line. Results： Four hybridomata secreting mAbs steadily were isolated successfully, and the serum titer of mAb in BALB/c mice was almost 10 times higher than that in five-feature mice. The indirect ELISA method for mAb titer determination was also established. The anti-VEGF165 mAb was purified to homogeneity by precipitation with ammonium sulfate followed by the affinity chromatography on MBP IgM purification column. Moreover, both purified human IgM V2, V75 and mouse ascites were characterized by SDS-PAGE and Western blotting. Proliferation of T24 cell line was considerably inhibited by V2 and V75. Conclusion： Five-feature mice could be used to produce fully human monoclonal antibody. The fully human anti-VEGF mAb is potential in the cancer treatment.